Non-melanoma skin cancers (NMSC), comprising squamous cell (SCC) and basal cell carcinomas (BCC), have a significant impact upon the health care system because their overall incidence is higher than for all other cancers combined. Therefore, while not usually fatal, these skin carcinomas incur a very high cost for their management, e.g., 90% of the cost of treating breast cancer. The current standard of care is surgical excision with wide margins. Major morbidity stems from fibrosis, scarring, and loss of function after treatment. Photodynamic therapy (PDT) using 5-aminotevulinicacid (ALA) represents a non-disfiguring alternative to surgery, especially for patients with multiple tumors and for tumors in delicate sites. Currently, the efficacy of ALA-PDT is not adequate to realistically compete with surgery in the U.S.A. Based on one of the Program themes of Combination Photodynamic Biologic Therapy (CPBT), in Project 1, our overall hypothesis is that tumor-differentiating agents can be used to improve the efficacy of ALA-PDT so that PDT becomes a viable alternative to surgery for NMSC. Wewill build upon our discovery that two small molecules, methotrexate (MTX) and vitamin D (Vit D), can increase tumor cell differentiation and at the same time increase the levels of photosensitizer (PplX) within the cells, thus enhancing responsiveness to therapy. The project is translational in nature, with clinical and basic science components. Aim 1 is preclinical, and uses mouse models of SCC and BCC to determine optimal dosing regimens for the systemic differentiating agent. Aims 2 and 3 are clinical studies to determine the efficacy of topical Vit D and oral MTXas combination agents with ALA-PDT. These Aims will also test new in vivo multimodal subsurface imaging devices for PplX detection, and evaluate C/EBP transcription factors ex vivo as prognostic markers of tumor response. Aim 4 consists of mechanistic experiments that will examine regulatory functions and the prognostic value of the C/EBPs in terms of PplX accumulation, using the preclinical models and examining tissue-banked skin tumor specimens from the clinical studies. Aim 4 is significant because those experiments will contribute to further improvements in the treatment design. The project involves significant collaborations with Project 4, Core B, and Core C of the Program. In summary, the potentialbenefits to public health will be the development of new rational combination approaches for PDT of skin cancer that include the manipulation of tumor cell physiology to increase endogenous levels of PplX. This will be coupled with individualized treatment planning based upon advanced PplX dosimetry and measurement of differentiation-responsive molecular markers, to optimize treatment response.